On-line electrochemical tagging of free cysteines in peptides during nanospray ionisation mass spectrometry: An overview

A recently developed nanospray interface for mass spectrometry (MS) was used to electro-generate benzoquinone from hydroquinone. When spraying biomolecules containing free cysteine residues in the presence of hydroquinone, the thiol cysteine moiety reacts via a 1,4-Michael addition with benzoquinone electrochemically generated at the electrode. The on-line labelling of cysteine was optimised using several substituted hydroquinones, and the kinetics of the addition was investigated by electrochemical methods such as cyclic voltammetry (CV). The mass spectrometric analysis of peptides containing up to three cysteines residues in the presence of selected probes allows the direct quantification of free cysteine residues. The on-line tagging method provides both the mass of the untagged peptides and that of the tagged peptides.